MUC2polymorphisms are associated with endometriosis development and infertility: a case-control study

Background: Mucins are highly glycosylated proteins protecting and lubricating epithelial surface of respiratory, gastrointestinal and reproductive tracts. Members of the mucin protein family have been suggested to play an important role in development of endometriosis and infertility. This study investigates genetic association of mucin2 ( MUC2) with the risk of endometriosis and endometriosis-related infertility.

This case-control study was conducted at China Medical University Hospital, with 195 endometriosis patients and 196 healthy controls enrolled. Genotyping of six SNPs (rs2856111, rs11245936, rs10794288, rs10902088, rs7103978 and rs11245954) within MUC2 gene were performed by using Taqman genotyping assay; individual SNP and haplotype associations with endometriosis and endometriosis-related infertility were assessed by c 2 test.

Endometriosis patients exhibit significantly lower frequency of the rs10794288 C allele, the rs10902088 T allele and the rs7103978 G allele ( P = 0.030, 0.013 and 0.040, respectively). In addition, the rs10794288 C allele and the rs10902088 T allele were also less abundant in patients with infertility versus fertile ones ( P = 0.015 and 0.024, respectively). Haplotype analysis of the endometriosis associated SNPs in MUC2 also showed significantly association between the most common haplotypes and endometriosis or endometriosis-related infertility.

MUC2 polymorphisms, especially rs10794288 and rs10902088, are associated with endometriosis as well as endometriosis-related infertility. Our data present MUC2 as a new candidate involved in development of endometriosis and related infertility in Taiwanese Han women.

Endometriosis is a common chronic gynecologic disease defined as presence of endometrial tissue outside the uterine cavity, primarily on pelvic peritoneum and ovary. Epidemiology studies reveal that endometriosis affects more than 10% of reproductive age women and possibly causes infertility [1,2]. The prevalence of endo- metriosis was 0.5-5% in fertile and 25-45% in infertile women [3]. The mechanism underlying endometriosis development remains unclear, even though theories like implantation, altered immuni ty, and susceptible genetic factors have been proposed to explain the pathogenesis [4-6]. Nevertheless, familial and identical twins studies have established the genetic predisposition to endome- triosis development [7]. Clinical manifestation of endometriosis is accompa- nied by angiogenesis and formation of cellular adhesion [8,9], possibly due to altered peritoneal environment and immune system [10]. In endometriosis patients, changes in levels of growth factors, cytokines and oncofetal anti- gens may facilitate intraperitoneal endometrial growth and alter the peritoneal environment, which leads to dis- ruption of normal pelvic organ architectures and inferti- lity [8]. For instance, interluekin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), nuclear factor- /C20B( N F -/C20B) and tumor necrosis factor- alpha (TNF- a) are among the major cytokines partici- pating in regulation of immune system, angiogenesis, * Correspondence: [email protected]; [email protected] † Contributed equally 3Human Genetics Center, China Medical University Hospital, 2 Yude Road, 40447 Taichung, Taiwan Full list of author information is available at the end of the article Chang et al . BMC Medical Genetics 2012, 13:15 http://www.biomedcentral.com/1471-2350/13/15 © 2012 Chang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cell proliferation and tissue invasiveness during the for- mation of endometriosis [11-14]. Mucins are high molecular weight glycoproteins with function of protecting and lubricating epithelial surface of respiratory, gastrointest inal and reproductive tracts [15]. Mucins are also found to be expressed on activated lymphocytes, supporting the hypothesis that some mucin domains function as cytokines to mediate immune responses [16,17]. Early studies reported that mucin played important role in the progress of tumor invasion, which is influenced by their glycosylation sta- tus [15,18-20]. More recently, mucin1 (MUC1) has been reported to be linked to endometriosis and infertility [21,22], and mucin4 (MUC4 ) gene polymorphisms were proved to be associated with endometriosis development as well [23]. The human mucin2 ( MUC2 )g e n ei s located on chromosome 11p15.5, encoding one of the most common gel-forming secreted type of mucin [24]. MUC2 expression was reported to be regulated by many endometriosis-related cytokines, such as IL-1 b,I L - 6 , TNF- a, NF-Kappa B [9,25-27]. Abnormal increase of mucin2 (MUC2) expression was reported to be linked to intestinal and uterine ce rvix metaplasia progression [28,29]. However, there has been no study yet investigating the relationship between endometriosis and MUC2, while previous functional studies on MUC2 are more focused on its role in the gastrointestinal and respiratory tract [30,31]. The aim of this study was to investigate the possible association of MUC2 gene polymorphisms with the risk of endometriosis and endometriosis-related clinical symptoms in a Taiwanese population.

Subjects In all, 195 patients receiving surgery for ovarian benign disease and pathology-prov en endometriosis patients were identified at China Medical University Hospital from 1998-2011 and enrolled in the study. They accepted examination of ultr asound before operation and were diagnosed with ovarian cysts; symptoms of dysmenorrhea, lower abdominal pain, infertility or abnormal menstruation were recorded for each patient. Another 196 healthy controls were recruited from a pool of persons who received regular health checkups at the same hospital. People with ovarian cysts detected by ultrasound or any symptoms of dysmenorrhea, lower abdominal pain, infertility, abnormal menstruation were excluded from the controls. Both patients and controls share similar age profile. Clinical information of patients was collected from medical charts, including clinical stage, lesion size, location, drug treatment and fertility. The definition of endometriosis staging was based on criteria of the American Society for Reproductive Medi- cine: stage 1, minimal; stage 2, mild; stage 3, moderate; and stage 4, severe [32]. Individual leukocytes were drawn by vein-puncture and separated by Ficoll-paque (GE, Uppsala, Sweden) for genomic DNA isolation. Signed consent was obtained from each study subject, approved by the Institutional Review Board at China Medical University Hospital. Genotyping Genomic DNA was extracted from peripheral blood leu- kocytes according to standard protocol (Genomic DNA kit; Qiagen, Valencia, CA, USA). DNA fragments con- taining target SNP sites were amplified by PCR using the Taqman SNP genotyping assay system from Applied B i o s y s t e m s ,I n c .( C a r l s b a d ,C A ,U S A ) .T h ep r o b eI D s for these SNP sites were selected from the ABI SNP genotyping databank http://www.allsnps.com and listed in Additional file 1: Table S1. PCR amplification condi- tions consisted of initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, and 56°C for 10 sec. Genetic variations were detected by reading the fluorescence signals of PCR products. A positive fluores- cent signal indicates a perfect match between the probe and the tested DNA, thus identifying the allele type. Statistical analysis Allelic and genotypic frequency distributions for these SNP sites in endometriosis patients and controls were performed by c 2 analysis using SPSS software (version 10.0, SPSS Inc. Chicago, Illinois, US). Allelic and genoty- pic frequencies are expressed as percentages of the total number of alleles and genotypes. Odds Ratios (ORs) were calculated for allelic and genotypic frequencies with 95% confident interval (95% CI). Adherence to the Hardy-Weinberg equilibrium constant was confirmed by c 2 test with one degree of freedom by PLINK program [33]. Haplotype association was analyzed using Bayesian statistical method available in the program Phase 2.1 [34]. Lewontin ’s coefficient D ’ and the linkage disequili- brium (LD) were determined between each pairs of bial- lelic loci using absolute association ( r 2) [35]. Haploview 4.2 (Whitehead Institute for Biomedical Research, Cam- bridge, MA) was used to examine the structure of the LD block [36]. A p v a l u eo fl e s st h a n0 . 0 5w a sc o n s i d - ered statistically significant. Functional analyses and secondary structure prediction Functional characterization and annotation of MUC2 were performed by aligning the sequence with func- tional motifs in PROSITE protein domain database [37]. NetSurfP ver. 1.1 was used to predict the secondary structure and surface accessibility of MUC2 [38] Relative Chang et al . BMC Medical Genetics 2012, 13:15 http://www.biomedcentral.com/1471-2350/13/15 Page 2 of 7 and absolute surface accessibility were also calculated for each residue.

MUC2 polymorphisms and endometriosis To test whether SNPs in MUC2 gene play role in endo- metriosis development, a total of six SNPs were selected for this study, with minor allelic frequencies over 4% in Chinese Han population based on the information in international HapMap project databank http://www.hap- map.org (Additional file 1: Table S1). These SNPs and t h e i rL Dm a pw e r es h o w ni nF i g u r e1 A .G e n o t y p ea n d allele frequencies were summarized in Table 1 for both the patient and the control g roups. Allele distribution analyses revealed significant associations between endo- metriosis and genetic variations at three of the six SNPs (Table 1). Endometriosis patients had significantly lower frequencies of C allele at rs10794288, T allele at rs10902088 and G allele at rs7103978, as compared to the controls ( P = 0.030, 0.013 and 0.040, respectively) (Table 1). Genotype analysis indicated that frequency of the TT genotype at rs10902088 was significantly lower in patients than in controls ( P = 0.045; OR = 0.47, 95% CI: 0.27-0.83), while the CC genotype at rs10794288 and the AG genotype at rs7103978 had protective effect against endometriosis (OR = 0.56 and 0.47, respectively; 95% CI < 1) (Table 1). These results implied possible relationship between three individual MUC2 polymorph- isms and endometriosis development. MUC2 polymorphisms and infertility Because endometriosis was suggested as one source of female infertility, we asked whether genetic variations in MUC2 play roles in this process. The patients were sub- grouped into patients with infertility or without inferti- lity for genotype and allele distribution analyses. Patients without sexual experience were excluded in this study. As shown in Table 2, the C allele at rs10794288 and the T allele at rs10902088 were much less prevalent in patients with infertility ( P = 0.015 and 0.024, respec- tively). The TT genotype at rs10902088 were absent in the infertile patient group , and genotype distribution was also significantly different between the infertile and the fertile patients for this SNP ( P = 0.047, data not shown). This finding revealed protective potential of these two genetic variations in MUC2 against infertility in endometriosis patients. Haplotype analysis of MUC2 polymorphisms The endometriosis-related SNPs (rs10794288, rs10902088 and rs7103978) found in individual tests were selected for haplotype analysis of endometriosis and disease-related clinical symptoms. Association data were enlisted in Table 3 for all the haplotypes with fre- quency higher than 1% presented in the case or control group. As shown in Table 3, the two most common haplotypes (T-C and C-T) of rs10794288 and rs10902088 were significantly associated with both endometriosis and infertility development in the patients. Haplotype T-C was more common in endome- triosis patients ( P = 0.012) and patients with infertility (P = 0.0091) (Table 3). By contrast, haplotype C-T might be a protective factor against endometriosis ( P = 0.035) and endometriosis-related infertility ( P = 0.025) (Table 3). Similarly, the most frequent haplotype T-C-A of rs10794288, rs10902088 and rs7103978 was signifi- cantly associated with both endometriosis ( P = 0.0063) and endometriosis-related infertility ( P = 0.0066) as a risk factor of the disease (Table 3). In the patient group, L Dm a pd e m o n s t r a t e dh i g h e rl i n k a g eb e t w e e nt h e s e three SNPs (Figure 1B) compared to the control group (Figure 1A), suggesting a genetic relationship between these SNPs and endometriosis. Therefore, haplotypes of these SNPs in MUC2 gene could serve as an indicator of susceptibility to endometriosis and endometriosis- related infertility. MUC2 polymorphisms and amino acid substitutions One of the endometriosis-associated SNPs (rs1090 2088) in MUC2 gene caused an amino acid substitu- tion (Additional file 1: Ta ble S1), and functions of MUC2 might be altered if such substitution changes surface charge, protein stability or folding. This SNP located at amino acid Asn1149, which was predicted to be within a long coil region. The relative surface acces- sibility of Asn1149 was estimated to be 0.707 with

to a fully exposed side chain, representing a highly exposed residue. Genetic variation of rs10902088 introduced an asparagine to lysine substi- tution, which was well fit in this position as a highly exposed residue, but the surface charge at this region would be reversed. Accordingly, this substitution was A B Figure 1 Pairwise linkage disequilibrium (LD) between SNPs of the MUC2 gene . LD maps were shown for controls (A) and patients (B). Values shown are for D ’. Chang et al . BMC Medical Genetics 2012, 13:15 http://www.biomedcentral.com/1471-2350/13/15 Page 3 of 7 more likely to disrupt inter- or intra-molecular interac- tions of MUC2 rather than protein stability. On the other hand, rs10794288 and rs7103978 were silent mutations, which only influence codon usages instead of amino acid substitutions.

Mucin proteins were known to be heavily glycosylated, on which oligosaccharide structures turned to be tumor-associated antigens and are essential for antibody recognition [39-41]. Our data revealed two MUC2 poly- morphisms (rs10794288 and rs10902088) were asso- ciated with endometriosis development and the related infertility. Polymorphism at rs10902088 generates an amino-acid change Asn1149Lys, while rs10794288 is a silent substitution. Altho ugh Asn1149 is not a typical site for N-linked glycosylation, this substitution to posi- tively charged residue may influence the glycosylation states of several neighboring serines and Asn1154, which is within a typical N-li nked glycosylation tripep- tide sequon Asn-Ile-Ser [42]. Therefore, this polymorph- ism may alter the glycosylation status of MUC2, which may subsequently influence the interaction between MUC2 and host environments. Table 1 Association between SNPs in MUC2 gene and endometriosis in Taiwanese patients and controls SNP Genotype/allele No. (%) of patients No. (%) of controls p-valuea OR 95% CI rs2856111 CC 35 (18.5) 35 (18.2) 0.29 0.79 0.46-1.36 CT 96 (50.8) 111 (57.8) 0.69 0.43-1.10 TT 58 (30.7) 46 (24.0) 1.00 C 166 (43.9) 181 (47.1) 0.37 0.88 0.66-1.17 T 212 (56.1) 203 (52.9) 1.00 rs11245936 AA 1 (0.5) 3 (1.6) 0.34 0.32 0.03-3.10 AG 21 (11.0) 28 (14.5) 0.72 0.39-1.32 GG 169 (88.5) 162 (83.9) 1.00 A 23 (6.0) 34 (8.8) 0.14 0.66 0.38-1.22 G 359 (94.0) 352 (91.2) 1.00 rs10794288 CC 34 (17.4) 45 (23.0) 0.092 0.56 0.33-0.96 CT 87 (44.6) 96 (49.0) 0.67 0.43-1.06 TT 74 (37.9) 55 (28.0) 1.00 C 155 (39.7) 186 (47.4) 0.030* 0.73 0.55-0.97 T 235 (60.3) 206 (52.6) 1.00 rs10902088 TT 27 (14.0) 43 (22.3) 0.045* 0.47 0.27-0.83 CT 93 (48.2) 95 (49.2) 0.74 0.47-1.16 CC 73 (37.8) 55 (28.5) 1.00 T 147 (38.1) 181 (47.4) 0.013* 0.70 0.52-0.93 C 239 (61.9) 205 (52.6) 1.00 rs7103978 GG 2 (1.0) 1 (0.5) 0.053 1.84 0.16-21.58 AG 19 (9.8) 37 (18.9) 0.47 0.26-0.85 AA 172 (89.1) 158 (80.6) 1.00 G 23 (6.0) 39 (9.9) 0.040* 0.57 0.34-0.98 A 363 (94.0) 353 (90.1) 1.00 rs11245954 GG 0 (0.0) 0 (0.0) NA NA NA AG 21 (10.8) 26 (13.5) 0.78 0.42-1.43 AA 173 (89.2) 166 (86.5) 1.00 G 21 (5.4) 26 (6.8) 0.43 0.79 0.44-1.43 A 367 (94.6) 358 (93.2) 1.00 *Indicates statistical significance aP-values were calculated using c2 test without corrections for multiple test Table 2 Association between allele distributions of SNPs in MUC2 and endometriosis-related infertility SNP Infertile MAF Non-Infertile MAF P-valuea OR 95% CI rs2856111 48.1 41.3 0.36 1.32 0.73-2.38 rs11245936 3.8 5.4 0.64 0.70 0.16-3.16 rs10794288 24.1 41.7 0.015* 0.44 0.23-0.87 rs10902088 26.0 43.1 0.024* 0.46 0.24-0.91 rs7103978 5.8 5.8 0.99 0.99 0.28-3.54 rs11245954 3.8 4.7 0.79 0.82 0.18-3.72 *Indicates statistical significance MAF minor allele frequency aP-values were calculated using c2 test Chang et al . BMC Medical Genetics 2012, 13:15 http://www.biomedcentral.com/1471-2350/13/15 Page 4 of 7 Endometriosis development is associated with altered inflammatory and immune responses, while clinical fea- ture of endometriosis also mimics malignant reproduc- tive disease, such as progressive invasion to adherent pelvic organ and recurrence abilities. Mucins are secreted by epithelium cells of reproductive tissues, gen- erating the mucus of cervix and endometrium, which plays an important role in reproductive physiology. Impaired mucin secretion could impede spermatozoa migration, which may contribute to female infertility. Unlike the less consistent expression patterns of MUC1, MUC6 and MUC5AC in normal and cancer tissues, MUC2 levels were always measured low in normal endometrial and cervical tissue, and elevated MUC2 expressions were specifically found in various neoplastic lesions [28,29,43-45]. However, expression patterns of MUC2 in ovarian tumor were heterogenic [18]. Boman et al. reported that MUC2 were mainly present in benign and borderline ovarian tumor [46], while Dong et al. showed that breast cancer patients with presence of MUC2 expression had short er disease-free survival [47]. We found that the minor allele of rs7103978 decreases the cognate co don frequency from 15.8 ‰ to 7.8‰ (Kazusa DNA Res. Inst. http://www.kazusa.or.jp/ codon/), which may increase the odds of premature translation termination and thus reduce MUC2 level. Therefore, our result suggested that expression of MUC2 may facilitate cell invasion or proliferation abil- ities. The observed association of MUC2 polymorphisms and endometriosis may help us further elucidate the link between endometriosis and certain subtypes of ovarian cancer, if such genetic alterations were also pre- sent in the ovarian cancer patients. Although endometriosis could cause pelvic adhesion and tubal occlusion which lead to infertility, some patients without anatomic disruption still had the pro- blem of impaired fertilizati on. Possible mechanisms of endometriosis-related infertility include impaired follicu- logenesis induced by abnormal immunological, chemical factors or toxins, poor oocyte quality, inhibited binding of spermatozoa to the zona-pellucida and impaired implantation of embryo. This phenomenon was corre- lated to changes of cytokines and growth factors in endometrium, follicular fluid and peritoneal fluid [8]. Previous studies have alread y showed the positive asso- ciation of endometriosis and polymorphism of cytokine genes [7,48]. Li et al. showed that macrophage induced IL-6 up-regulated the MUC1 but down-regulated MUC2 expression [9]. Up-regulation of MUC1 was associated with implantation failure [22]. IL-1 was also found to up-regulate MUC2 expression, and IL-1 was thought to regulate immune and inflammation response in endo- metrium and modulate extracellular matrix modeling of endometrium during menstruation and implantation [49]. Moreover, previous studies demonstrated that IL-8, TNF-a and NF- /C20B tend to increase in the peritoneal fluid according to the severity of dysmenorrhea, extent pelvic adhesion and proliferation of endometrial stroma cells, and MUC2 expression could increase accordingly via activation of NF- /C20B pathway through these cytokines [50-52]. Therefore, it is plausible that altered level of MUC2 could affect fertilit y as a downstream effecter that can further influence the secretion of mucus, sperm motility, oocyte quality and receptivity of endometrium.

In this study, our data revealed a significant association between MUC2 polymorphisms and endometriosis in a Taiwanese population. Th er e s u l t si m p l yt h a tM U C 2 may play a role in the pathogenesis of endometriosis and endometriosis-related infertility, while the mechan- isms underlying this phenomenon remain to be eluci- dated. As a major secreted form of mucins, MUC2 may have the ability to affect more surrounding tissues than the membrane-bound form of mucins. Since MUC2 is not as well investigated as some other mucins in repro- ductive organs, its molecular function in endometriosis and infertility is worth future study. Table 3 Association between MUC2 haplotypes and endometriosis or endometriosis-related infertility SNP marker Haplotype Endometriosis Endometriosis-related infertility Case (%) Control (%) P-valuea Infertility (%) Non-infertility (%) P-valuea rs10794288, rs10902088 T-C 58.6 49.6 0.012* 74.9 57.2 0.0091* C-T 35.8 43.2 0.035* 23.4 38.3 0.025* C-C 3.4 3.7 0.83 0.1 2.6 0.20 T-T 2.1 3.5 0.27 1.6 1.9 0.89 rs10794288, rs10902088, rs7103978 T-C-A 57.8 48.1 0.0063* 74.8 56.3 0.0066* C-T-A 32.0 35.9 0.25 22.0 34.1 0.06 C-T-G 3.8 7.2 0.036* 1.4 4.2 0.29 C-C-A 3.4 3.7 0.81 0 2.6 0.20 T-T-G 1.5 2.0 0.61 1.6 1.1 0.76 *Indicates statistical significance. aP-values were calculated using c2 test Chang et al . BMC Medical Genetics 2012, 13:15 http://www.biomedcentral.com/1471-2350/13/15 Page 5 of 7 Additional material Additional file 1: Table S1 . Probes been used for SNPs in MUC2 gene.

We appreciate the kind assistance from Ms Carmen Chan at China Medical University Hospital. This work was supported by the grant from China Medical University Hospital (DMR-99-091) and the China Medical University- Asia University Research Collaboration Fund (CMU98-asia-02), Taichung, Taiwan. Author details 1Department of Obstetrics and Gynecology, China Medical University Hospital, 2 Yude Road, 40402 Taichung, Taiwan. 2Institute of Public Health, China Medical University, 91 Hsueh-Shih Road, 40447 Taichung, Taiwan. 3Human Genetics Center, China Medical University Hospital, 2 Yude Road, 40447 Taichung, Taiwan. 4Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92 Sec. 2 Zhongshan Road, 10449 Taipei, Taiwan. 5School of Medical Imaging and Radiological Sciences, Chung Shan Medical University, 110 Sec.1 Jianguo N. Rd, 40201 Taichung, Taiwan. 6School of Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, 40447 Taichung, Taiwan. 7Department of Health and Nutrition Biotechnology, Asia University, 500 Lioufeng Road, 41354 Taichung, Taiwan. 8School of Post- Baccalaureate Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, 40447 Taichung, Taiwan. Authors’ contributions CY-YC and YC: study design, execution and manuscript drafting; YC and C- MC: statistical analysis; CY-YC, W-CL and F-JT: patient collection; C-PC, S-CL and JJ-CS: critical discussion and manuscript editing. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 17 October 2011 Accepted: 15 March 2012 Published: 15 March 2012

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